Synthetic analogues of neural regeneration peptides

ABSTRACT

Embodiments of this invention include synthetic compounds (NRP analogues) of peptides termed neural regeneration peptides (NRPs). NRP analogues are made by substituting amino acids in the native peptide sequence, modifying amino acids chemically, by replacing amino acids with synthetic moieties, by stabilizing β-turns, acetylation of terminal glycine residues or by cyclization. NRP analogues can be used to treat a variety of conditions involving degeneration of neural cells, and includes treating disorders of the nervous system, including peripheral neuropathy, multiple sclerosis, diabetic peripheral neuropathy, neurotoxin-induced neurodegeneration, and amyotrophic lateral sclerosis.

CLAIM OF PRIORITY

This United States Continuation Patent Application claims priority toPCT International application No PCT/US2008/011951, filed 17 Oct. 2008,entitled “Synthetic Analogues of Neural Regeneration Peptides,” whichclaims priority to U.S. Provisional Application No: 60/999,292 entitled“Synthetic Analogs of Neural Regeneration. Peptides,” filed Oct. 17,2007, and to U.S. Provisional Patent Application No: 60/999,503,entitled “Synthetic Analogs of Neural Regeneration Peptides,” filed Oct.18, 2007. Each of these applications are incorporated herein fully byreference as if individually so incorporated.

FIELD OF THE INVENTION

This invention relates to synthetic analogues of peptides that haveneural regeneration, migration, proliferation, differentiation and/oraxonal outgrowth properties. These peptides are termed “NeuralRegeneration Peptides” or “NRPs.” In particular, this invention relatesto analogues of relatively small peptides that have one or morebiological properties of NRPs.

BACKGROUND

Neural regeneration peptides (NRPs) are a class of peptides that havebeen shown to exhibit properties desirable for promoting neural functionin mammals. These functions include neural survival, neuralproliferation, neuronal outgrowth, neural migration and neuronaldifferentiation. Several NRPs have been previously described, andinclude those disclosed in U.S. patent application Ser. Nos. 10/225,838and 10/976,699, PCT/US02/026782, PCT/US2004/036203, PCT/US2006017534 andPCT/US2006026994. Each of the above patent applications is expresslyincorporated herein fully by reference as if individually soincorporated.

SUMMARY

NRPs described to date have desirable pharmacodynamic properties andpromote neural regeneration, migration, proliferation, differentiationand/or axonal outgrowth. We have recently discovered synthetic NRPanalogues that also have improved pharmacokinetic properties. There is aneed in the art for synthetic molecules or modified peptides that havedesirable pharmacodynamic properties similar to those of NRPs but alsohave improved pharmacokinetic properties and/or are chemically stable.

Certain aspects of this invention include novel synthetic NRP analoguemolecules that can be used to treat disorders of the nervous system orother systems in which NRPs is effective. Another aspect is to providetherapies for disorders of cellular degeneration and death, includingcertain nervous system disorders. In some aspects, synthetic analoguesof NRPs can be used to treat adverse effects of amyotrophic lateralsclerosis (ALS), multiple sclerosis (MS), oxidative stress (e.g.,Huntington's disease) or peripheral neuropathy (PN). An additionalaspect of this invention is the production of NRP analogues withimproved stability.

It should be understood that the terms “NRP compound,” “analogue of NRP”“SEQ ID NO:” and other such terms, for simplicity, are used to identifythe molecules of the invention and not to provide their completecharacterization. Thus, an “analogue of NRP” may be characterized hereinas having a particular amino acid sequence, a particular 2-dimensionalrepresentation of the structure, but it is understood that the actualmolecule claimed has other features, including 3-dimensional structure,mobility about certain bonds and other properties of the molecule as awhole. It is the molecules themselves and their properties as a wholethat are the subjects of this invention.

It should also be understood that the designation of a peptide as an“NRP” does not mean that it solely has neural effects. Rather, the termNRP is intended to include peptides having similar structural componentsas described in the above patent applications, but may have effects onother cell types, tissues, and/or organs. In certain embodiments,analogues of relatively short NRPs are provided that can have increasedstability, due at least in part to decreased enzymatic degradation. Inother embodiments, NRP analogues are provided having modified aminoacids. In yet further embodiments, NRP analogues are provided that havenon-amino acid substituents replacing amino acids.

BRIEF DESCRIPTION OF THE FIGURES

This invention will be described with reference to specific embodimentsthereof. Other features and aspects of this invention can be appreciatedby reference to the Figures, in which:

FIG. 1 depicts a graph of neuroprotective effects of two NRPs, SEQ IDNO: 5 of this invention and SEQ ID NO:1 in cell cultures exposed to theneurotoxin 3-NP.

FIG. 2 depicts a graph of results of studies of neuroprotectiveeffective of SEQ ID NO:1 in which the peptide was stored at either −20°C. or −4° C.

FIG. 3 depicts a graph of results of studies of neuroprotective effectsof SEQ ID NO: 5 of this invention in which the peptide was stored ateither −20° C. or −4° C.

FIG. 4 depicts a graph of results of an enlarged study ofneuroprotective effects of SEQ ID NO:5 of this invention and SEQ ID NO:1in cell cultures treated with the neurotoxin 3-NP, similar to thoseshown in FIG. 1.

FIG. 5 depicts a graph showing significant long-term effects of sequenceREGRRDAPGRAGG (SEQ ID NO:12) of this invention to decrease the severityof motoric impairment in animals with EAE, when the synthetic NRP wasadministered at the peak of the disease. Score 1 is the lowest score andimplies a flaccid tail only, while the higher scores imply weakness(score 2) or complete paralysis of the hind legs (score 3).Kruskal-Wallis-test was used for statistical analysis; **p<0.01 versustreatment day 1 score (data expressed as mean±SEM).

FIG. 6 depicts a graph of results of beam walking scores for rats withperipheral neuropathy induced by pyridoxine (800 mg/kg/day) and treatedwith either vehicle or SEQ ID NO:5 of this invention at two differentdoses.

FIG. 7 depicts a graph of results of beam walking scores for rats withperipheral neuropathy induced by pyridoxine (1200 mg/kg) and treatedwith either vehicle or SEQ ID NO:5 of this invention.

FIG. 8 depicts graphs of results of studies of longevity of mice with amurine model of amytrophic lateral sclerosis (ALS) and the effects oftwo different doses of a synthetic NRP of this invention, SEQ ID NO:5.

DETAILED DESCRIPTION

In some embodiments, NRP compounds are provided that have a sequence ofa native peptide. For example, one such NRP is an 11 amino acid long(11-mer) peptide having the following sequence.

NH₂-G¹RRAAPGRAGG¹¹-NH₂ SEQ ID NO: 1

It should be appreciated that synthetic compounds or analogues of NRPscan have either amidated C-termini or can have C-terminal hydroxylresidues (OH). It should also be appreciated that the terms “NRPcompound,” “NRP analogue” and similar terms refer to compounds of thisinvention or to previously disclosed NRP peptides or NRP proteins.

Synthetic Analogues of NRPs

Synthetic analogues of NRPs are provided that can have one or more ofthe following types of modifications: (1) stabilization of β-turns, (2)replacement of glycine residues, (3) replacement of the N-terminalglycine residue and/or (4) cyclization.

1. Stabilization of β-Turns

Chou and Fasman probabilities for β-turn prediction reveal that probableβ-turns in SEQ ID NO:1 can be found in the domains “APGR (SEQ ID NO:2)”and “RAGG (SEQ ID NO:3)”, as shown in bold below:

SEQ ID NO: 1 SEQ ID NO: 1. NH₂-G¹RRAAPGRAGG¹¹-NH₂

β-turns can be stabilised by introducing steric constraints such asalkylated amino acids. Readily available amino acids that can be usedinclude aminoisobutyric acid (Aib, α-H on alanine replaced with methyl)can be used as a replacement for either or both of alanine and glycineresidues.

A. Modification of the APGR Domain

In sequence APGR (SEQ ID NO: 2), the alanine or glycine can be replacedwith aminoisobutyric acid (Aib). Substitution of the alanine with Aibproduces the following analogue:

NH₂-G¹RRA-Aib-PGRAGG¹¹-NH₂ SEQ ID NO: 4

B. Modification of the RAGG Domain

In another probable β-turn sequence, RAGG (SEQ ID NO: 3), the alaninecan be replaced with aminoisobutyric acid (Aib) to produce the analoguehaving the sequence

NH₂-G¹RRAAPGR-Aib-GG¹¹-NH₂ SEQ ID NO: 5

Experiments showed that SEQ ID NO:5 was: neuroprotective (see FIGS.1-4), stable under storage conditions (FIGS. 2 and 3), moreneuroprotective than the unsubstituted NRP (FIGS. 1 and 4), peripheralneuropathy (FIGS. 6 and 7) and ALS (FIG. 8).

2. Replacement of Glycine Residues

Replacement of the internal glycine residue by an asparagine (N) caninduce β-turns due to asparagine having higher β-turn propensity thanglycine. Therefore the internal glycine residue can be replaced withasparagine at amino acid position 10 producing a peptide having thefollowing sequence:

NH₂-G¹RRAAPGRANG¹¹-NH₂ SEQ ID NO: 6

This NRP analogue was found to be neuroprotective in vitro model ofneural toxicity induced by 3-NP.

3. Replacement of the N-Terminal Glycine Residue

Truncation of the G¹ at the N terminus can result in loss of biologicalactivity. Replacement of the G¹ with an acetyl group can restorebiological activity. The resulting NRP analogue is acetylated providinga peptide having the following sequence:

AcNH-RRAAPGRAGG¹¹-NH₂ SEQ ID NO: 7

This NRP analogue was found to be neuroprotective in the face oftoxicity induced by 3-NP.

4. Replacement of L-Amino Acids with D-Amino Acids

The secondary structure of a peptide can be affected by the presence ofD-amino acids replacing one or more L-amino acids (naturally occurring).Replacement of the third amino acid from the N-terminus produces acompound having the following sequence:

NH₂-GR (D-Arg) AAPGRAGG-NH₂ SEQ ID NO: 8

This NRP analogue was found to be neuroprotective in the in vitro modelof neural toxicity induced by 3-NP.

5. Cyclization

Synthesis of a cyclic peptide mimetic of SEQ ID NO:1 can be carried out.One method involves adding a cysteine residue to each end of thesequence, and then oxidizing the resultant product to produce a cyclicdisulfide having the following sequence:

Alternatively, both the N and C terminal glycine residues can bereplaced with a cysteine residue and oxidized similarly as above,producing an analogue having the following sequence.

Direct cyclization of the C terminal residue to the N terminal residuecan be accomplished by creating an amide bond to produce a peptidehaving the following sequence.

The use of circular dichroism can indicate secondary structure and theuse of computer simulation software for the modeling of small peptidescan also be carried out using conventional methods. Both of thesetechniques can be used for determining structural features of the NRPanalogues of this invention.

Synthesis of Synthetic Analogues of NRPs

Starting materials and reagents used in preparing these compounds areeither available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to the person of ordinaryskill in the art following procedures described in such references asFieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, JohnWiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of CarbonCompounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989;Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y.,1991; March J; Advanced Organic Chemistry, 4^(th) ed. John Wiley andSons, New York, N.Y., 1992; and Larock: Comprehensive OrganicTransformations, VCH Publishers, 1989. In most instances, amino acidsand their esters or amides, and protected amino acids, are widelycommercially available; and the preparation of modified amino acids andtheir amides or esters are extensively described in the chemical andbiochemical literature and thus well-known to persons of ordinary skillin the art. For example, N-pyrrolidineacetic acid is described inDega-Szafran Z and Pryzbylak R. Synthesis, IR, and NMR studies ofzwitterionic α-(1-pyrrolidine)alkanocarboxylic acids and their N-methylderivatives. J. Mot. Struct.: 436-7, 107-121, 1997; andN-piperidineacetic acid is described in Matsuda O, Ito S, and Sekiya M.each article herein expressly incorporated herein fully by reference.

Conveniently, synthetic production of the polypeptides of the inventionmay be according to the solid-phase synthetic method described byGoodman M. (ed.), “Synthesis of Peptides and Peptidomimetics” in Methodsof organic chemistry (Houben-Weyl) (Workbench Edition, E22a,b,c,d,e;2004; Georg Thieme Verlag, Stuttgart, N.Y.)., herein expresslyincorporated fully by reference. This technique is well understood andis a common method for preparation of peptides. The general concept ofthis method depends on attachment of the first amino acid of the chainto a solid polymer by a covalent bond. Succeeding protected amino acidsare added, on at a time (stepwise strategy), or in blocks (segmentstrategy), until the desired sequence is assembled. Finally, theprotected peptide is removed from the solid resin support and theprotecting groups are cleaved off. By this procedure, reagents andby-products are removed by filtration, thus eliminating the necessity ofpurifying intermediaries.

Amino acids may be attached to any suitable polymer as a resin. Theresin must contain a functional group to which the first protected aminoacid can be firmly linked by a covalent bond. Various polymers aresuitable for this purpose, such as cellulose, polyvinyl alcohol,polymethylmethacrylate and polystyrene. Suitable resins are commerciallyavailable and well known to those of skill in the art. Appropriateprotective groups usable in such synthesis include tert-butyloxycarbonyl(BOC), benzyl (Bzl), t-amyloxycarbonyl (Aoc), tosyl (Tos),o-bromo-phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BzlCl₂), andphenylmethoxycarbonyl (Z or CBZ). Additional protective groups areidentified in Goodman, cited above, as well as in McOmie J F W:Protective Groups in Organic Chemistry, Plenum Press, New York, 1973,both references expressly incorporated fully herein by reference.

General procedures for preparing peptides of this invention involveinitially attaching a carboxyl-terminal protected amino acid to theresin. After attachment the resin is filtered, washed and the protectinggroup on the alpha-amino group of the carboxyl-terminal amino acid isremoved. The removal of this protecting group must take place, ofcourse, without breaking the bond between that amino acid and the resin.The next amino, and if necessary, side chain protected amino acid, isthen coupled to the free amino group of the amino acid on the resin.This coupling takes place by the formation of an amide bond between thefree carboxyl group of the second amino acid and the amino group of thefirst amino acid attached to the resin. This sequence of events isrepeated with successive amino acids until all amino acids are attachedto the resin. Finally, the protected peptide is cleaved from the resinand the protecting groups removed to reveal the desired peptide. Thecleavage techniques used to separate the peptide from the resin and toremove the protecting groups depend upon the selection of resin andprotecting groups and are known to those familiar with the art ofpeptide synthesis.

Peptides may be cyclized by the formation of a disulfide bond betweentwo cysteine residues. Methods for the formation of such bonds are wellknown and include such methods as those described in G. A. Grant (Ed.)Synthetic Peptides A User's Guide 2^(nd) Ed., Oxford University Press,2002, W. C. Chan and P. D. White (Eds.) Fmoc Solid Phase Synthesis APractical Approach, Oxford University Press, 2000 and referencestherein.

Alternative techniques for peptide synthesis are described in Bodanszkyet al, Peptide Synthesis, 2nd ed, John Wiley and Sons, New York, 1976,expressly incorporated herein fully by reference. For example, thepeptides of the invention may also be synthesized using standardsolution peptide synthesis methodologies, involving either stepwise orblock coupling of amino acids or peptide fragments using chemical orenzymatic methods of amide bond formation (see, e.g. H. D. Jakubke inThe Peptides, Analysis, Synthesis, Biology, Academic Press, New York,1987, p. 103-165; J. D. Glass, ibid., pp. 167-184; and European Patent0324659 A2, describing enzymatic peptide synthesis methods.) Thesesolution synthesis methods are well known in the art.

Commercial peptide synthesizers, such as the Applied Biosystems Model430A, are available for the practice of these methods.

Therapeutic Uses of NRP Analogues

NRP analogues of this invention can be used to treat neurologicaldisorders. NRPs have been unexpectedly effective in treating neuraldegeneration associated with autoimmune disorders of the brain,including EAE and multiple sclerosis (MS), amyotrophic lateral sclerosis(ALS) and toxic injury to neural cells.

Disorders and Conditions Treatable with NRP Analogues

Disorders and conditions in which NRP compounds of this invention can beof benefit include the following.

Nervous system conditions treatable with NRP analogues includeinfections of the central nervous system including bacterial, fungal,spirochetal, parasitic and sarcoid including pyrogenic infections, acutebacterial meningitis or leptomeningitis.

Cerebrovascular diseases include stroke, ischemic stroke,hypoxidischemia, atherosclerotic thrombosis, lacunes, embolism,hypertensive haemorrhage, ruptured aneurysms, vascular malformations,transient ischemic attacks, intracranial haemorrhage, spontaneoussubarachnoid haemorrhage, hypertensive encephalopathy, inflammatorydiseases of the brain arteries, decreased perfusion caused by, forexample, cardiac insufficiency (possibly resulting from coronary bypasssurgery) and other forms of cerebrovascular disease.

Craniocerebral traumas include basal skull fractures and cranial nerveinjuries, carotid-cavernous fistula, pneumocephalus, aeroceleandrhinorrhea, cerebral contusion, traumatic intracerebral haemorrhage,traumatic brain injury, penetrating traumatic brain injury and acutebrain swelling in children.

Demyelinating diseases include neuromyelitis optica, acute disseminatedencephalomyelitis, acute and subacute necrotizing haemorrhagicencephalitis, diffuse cerebral sclerosis of Schilder and multiplesclerosis in conjunction with peripheral neuropathy. Degenerativediseases of the nervous system including syndrome of one or more ofprogressive dementia, diffuse cerebral atrophy, diffuse cortical atrophyof the non-Alzheimer type, Lewy body dementia, Pick's disease,fronto-temporal dementia, thalamic degeneration, non-Huntingtonian typesof Chorea and dementia, cortico-spinal degeneration (Jakob), thedementia-Parkinson-amyotrophic lateral sclerosis complex (Guamanina andothers) and amyotrophic lateral sclerosis (ALS).

Peripheral neuropathies are common and disabling conditionscharacterised by damage to or loss of peripheral neurons. There are morethan 100 types of peripheral neuropathy, each with its owncharacteristic set of symptoms, pattern of development, and prognosis.Peripheral neuropathy may be either inherited or acquired. Inheritedforms of peripheral neuropathy can be caused by genetic mutations. Sometypes of peripheral neuropathy and features common to them are shownbelow in Table 1. Table 1 above shows comparisons between pyridoxine-,streptozotin- and chemotherapy-induced peripheral neuropathy anddiabetic peripheral neuropathy.

TABLE 1 Features Common to Peripheral Neuropathies ChemotherapyPyrodoxine Streptozotocin- Diabetic Induced (Vitamin B6) InducedDiabetic Peripheral Peripheral Intoxication Neuropathy NeuropathyNeuropathy Model/ Experimental Experimental Clinical ConditionExperimental Condition Model & Clinical Model Model & Clinical ConditionCondition Principle Reversible Peripheral nerve Progressive Peripheralnerve Pathology peripheral nerve axonpathy, peripheral nerveneuronpathy, as axonopathy, resulting in axonopathy, well as damage toresulting in reduced autonomic resulting in dorsal root ganglia reducedsensory and sensory fiber reduced sensory resulting in fiber conductionconduction fiber conduction reduced sensory velocity, restrictedvelocity, with velocity, with fiber conduction to large diameterdegeneration degeneration velocity, with and cells, without being fiberlength being fiber length without demyelination. dependent. dependent.demyelination. [1, 2, 3, 4, 5, 6] [11, 12, 13] [19, 20, 21] [22, 23]Proposed Saturation of Selective toxicity Wallerian axonal (For platinumMechanism pyridoxal kinase to islet β-cells of degeneration compounds)of Toxicity in the liver may the pancreas resulting from Disturbance ofinhibit neuronal leading to a hyperglycemic metabolism and pyridoxalhyperglycemic neurotoxicity. axonal transport in phosphate, diabeticcondition, Raised glucose peripheral sensory altering neuronal withresulting favors metabolism nerves following metabolism. neurotoxicity,as through the polyol accumulation in Imparied neuronal in clinicalpathway that the DRG leads to metabolism diabetes. results inaxonopathy. leads to an [14, 15, 16] production of [22, 24] impoverishedoxidative stress. energy support [16, 19, 20] of the large axons. [2, 3]Peripheral Primarily large Primarily Primarily large Primarily largeNerves descending autonomic and descending descending Affected sensorynerves, sensory nerves, sensory nerves, sensory nerves, includingincluding peroneal including peroneal including peroneal and and othernerves and sural nerves peroneal and sural nerves that descending fromthat descend from sural nerves that descend from the sciatic. thesciatic. descend from the sciatic. [11, 13] [19, 20] the sciatic. [3, 6,7] [22, 23] Functional Motoric Predominantly Initially positive SensoryOutcomes impairment of positive symptoms of pain neuronopathy,hind-limbs due symptoms of or paresthesia, with diminshed to loss ofsensory hyperalgesia/ then progressing vibration feedback, allodynia. tonegative perception, particularly from [17, 18] symptoms withparesthesia, loss the hind limbs. loss of sensory of tendon reflex, [3,6, 7, 8, 9, 10] feedback, pain and, later, particularly from ataxia(motoric the feet. impairment). [19, 20] [22, 23]

Acquired peripheral neuropathy may result from: physical injury (trauma)to a nerve, tumors, toxins (including chemotherapy), autoimmuneresponses, nutritional deficiencies, alcoholism, vascular and metabolicdisorders (e.g. diabetic neuropathy). The HIV-associated peripheralneuropathy is a common side effect of drugs targeting the reversetranscriptase of the HIV virus. The symptoms of peripheral neuropathycan vary from temporary numbness, tingling, and pricking sensations,sensitivity to touch or muscle weakness, to more extreme symptoms suchas burning pain, muscle wasting, paralysis, organ or gland dysfunction.

The first report of a human sensory neuropathy induced by a high dose ofpyridoxine (vitamin B6) derives from Schaumberg et al., New Eng. J.Medicine 309:445-448, 1983. Daily intakes were in the 2000-6000 mg/dayrange over periods from 2 to 14 months. All patients displayed a“stocking-glove” sensory loss with numbness in hands, feet and anunstable gait.

Using rat models of peripheral neuropathy enables the examination ofneurological abnormalities induced by pyridoxine. For instance, 1,200 or800 mg/kg/day of pyridoxine administered to rats for 5-10 days resultsin necrosis of sensory neurons, especially affecting large diameterneurons in the sciatic nerve and the dorsal root ganglion (Xu et al.,Neurology 39:1077-1083, 1989).

Pyridoxine-induced peripheral neuropathy in animals is a recognizedsystem for studying effects of therapeutic agents. In particular, thissystem is predictive of effects of such agents on peripheral neuropathyin human beings.

Metabolic Disorders

Acquired metabolic disorders of the nervous system including metabolicdiseases presenting as a syndrome comprising one or more of confusion,stupor or coma-ischemia-hypoxia, hypoglycaemia, hyperglycemia,hypercapnia, hepatic failure and Reye syndrome, metabolic diseasespresenting as a progressive extrapyramidal syndrome, metabolic diseasespresenting as cerebellar ataxia, hyperthermia, celiac-sprue disease,metabolic diseases causing psychosis or dementia including Cushingdisease and steroid encephalopathy, thyroid psychosis and hypothyroidismand pancreatic encephalopathy. An example of a metabolic disorder thatcan result in neuropathy is pyridoxine excess described more fullybelow.

Diseases of the Nervous System Due to Nutritional Deficiency, Alcoholand Alcoholism

Disorders of the nervous system due to drugs and other chemical agentsinclude opiates and synthetic analgesics, sedative hypnotic drugs,stimulants, psychoactive drugs, bacterial toxins, plant poisons,venomous bites and stings, heavy metals, industrial toxins,anti-neoplastic and immunosuppressive agents, thalidomide,aminoglycoside antibiotics (ototoxicity) and penicillin derivatives(seizures), cardioprotective agents (beta-blockers, digitalisderivatives and amiodarone).

As illustrated by the preceding list, compositions and methods of theinvention can find use in the treatment of human neural injury anddisease. Still more generally, the compositions and methods of theinvention find use in the treatment of human patients suffering fromneural damage as the result of acute brain injury, including but notlimited to diffuse axonal injury, perinatal hypoxic-ischemic injury,traumatic brain injury, stroke, ischemic infarction, embolism, andhypertensive haemorrhage; exposure to CNS toxins, infections of thecentral nervous system, such as, bacterial meningitis; metabolicdiseases such as those involving hypoxic-ischemic encephalopathy,peripheral neuropathy, and glycogen storage diseases; or from chronicneural injury or neurodegenerative disease, including but not limited toMultiple Sclerosis, Lewy Body Dementia, Alzheimer's disease, Parkinson'sdisease and Huntington's disease. Patient's suffering from such diseasesor injuries may benefit greatly by a treatment protocol able to initiateneuronal proliferation and migration, as well as neurite outgrowth.

Still more generally, the invention has application in the induction ofneuronal and neuroblast migration into areas of damage following insultin the form of trauma, toxin exposure, asphyxia or hypoxia-ischemia.

Administration of NRP Analogues

NRP analogues can be used via direct administration to the patient. AnNRP analogue can be administered as part of a medicament orpharmaceutical preparation. This can involve combining an NRP analoguewith any pharmaceutically appropriate carrier, adjuvant or excipient.Additionally an NRP analogue can be used with other non-NRPneuroprotective, proliferative, or other agent. The selection of thecarrier, adjuvant or excipient can depend upon the route ofadministration to be employed.

The administration route can vary widely to suit a particular condition.An NRP analogue may be administered in different ways:intraperitoneally, intravenously or intracerebroventricularly. Theperipheral application may be a route of choice because then there is nodirect interference with the central nervous system.

Any peripheral route of administration known in the art can be employed.These can include parenteral routes for example injection into theperipheral circulation, subcutaneous, intraorbital, ophthalmic,intraspinal, intracisternal, topical, infusion (using eg. slow releasedevices or minipumps such as osmotic pumps or skin patches), implant,aerosol, inhalation, scarification, intraperitoneal, intracapsular,intramuscular, intranasal, oral, buccal, pulmonary, rectal or vaginal.The compositions can be formulated for parenteral administration tohumans or other mammals in therapeutically effective amounts (eg.amounts which eliminate or reduce the patient's pathological condition)to provide therapy for the neurological diseases described above.

One route of administration includes subcutaneous injection (e.g.,dissolved in 0.9% sodium chloride) and oral administration (e.g., in acapsule).

It will also be appreciated that it may on occasion be desirable todirectly administer NRP analogue to the CNS of the patient by anyappropriate route of administration. Examples include administration bylateral cerebroventricular injection or through a surgically insertedshunt into the lateral cerebral ventricle of the brain of the patient,into the cerebrospinal fluid or directly into an affected portion of apatient's brain.

Therapeutic Doses of NRP analogues

In some embodiments of this invention, methods for treating brain damagecomprise administering one or more NRP analogues in a dose range of fromabout 0.01 μg/kg body weight to about 100 μg/kg body weight. In otherembodiments, a dose of 1 μg/kg body weight to about 10 μg/kg body weightcan be useful. In further embodiments, a dose of an NRP can be in therange of about 0.01 μg/kg body weight to about 0.1 mg/kg.

In other embodiments, the determination of an effective amount of an NRPanalogue to be administered is within the skill of one of ordinary skillin the art, and will be routine to those persons skilled in the art. Incertain embodiments, the amount of an NRP analogue to be used can beestimated by in vitro studies using an assay system as described herein.The final amount of an NRP analogue to be administered will be dependentupon the route of administration, upon the NRP analogue used and thenature of the neurological disorder or condition that is to be treated.A suitable dose range may for example, be between about 0.01 μg to about15 μg per 1 kg of body weight or in other embodiments, about 20 μg/kg toabout 30 μg/kg body weight per day.

For inclusion in a medicament, NRP analogue can be directly synthesizedby conventional methods such as the stepwise solid phase synthesismethod of Merrifield et al., 1963 (J. Am. Chem. Soc. 15:2149-2154) orGoodman M. (ed.), “Synthesis of Peptides and Peptidomimetics” in Methodsof organic chemistry (Houben-Weyl) (Workbench Edition, E22a,b,c,d,e;2004; Georg Thieme Verlag, Stuttgart, N.Y.), expressly incorporatedherein fully by reference. Such methods of peptide synthesis are knownin the art, and are described, for example, in Fields and Colowick,1997, Solid Phase Peptide Synthesis (Methods in Enzymology, vol. 289),Academic Press, San Diego, Calif., expressly incorporated herein fullyby reference. Alternatively synthesis can involve the use ofcommercially available peptide synthesizers such as the AppliedBiosystems model 430A.

As a general proposition, the total pharmaceutically effective amount ofan NRP analogue administered parenterally per dose will be in a rangethat can be measured by a dose response curve. For example, an NRPanalogue in the blood can be measured in body fluids of the mammal to betreated to determine dosing. Alternatively, one can administerincreasing amounts of an NRP compound to the patient and check the serumlevels of the patient for the NRP analogue. The amount of NRP analogueto be employed can be calculated on a molar basis based on these serumlevels of the NRP analogue.

One method for determining appropriate dosing of the compound entailsmeasuring NRP levels in a biological fluid such as a body or bloodfluid. Measuring such levels can be done by any means, including RIA andELISA. After measuring NRP analogue levels, the fluid is contacted withthe compound using single or multiple doses. After this contacting step,the NRP analogue levels are re-measured in the fluid. If the fluid NRPanalogue levels have fallen by an amount sufficient to produce thedesired efficacy for which the molecule is to be administered, then thedose of the molecule can be adjusted to produce maximal efficacy. Thismethod can be carried out in vitro or in vivo. This method can becarried out in vivo, for example, after the fluid is extracted from amammal and the NRP analogue levels measured, the compound herein isadministered to the mammal using single or multiple doses (that is, thecontacting step is achieved by administration to a mammal) and then theNRP analogue levels are re-measured from fluid extracted from themammal.

NRP analogues are suitably administered by a sustained-release system.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, forexample, films, or microcapsules. Sustained-release matrices includepolylactides (U.S. Pat. No. 3,773,919, EP 58,481), poly(2-hydroxyethylmethacrylate) (Langer et al., 1981), ethylene vinyl acetate (Langer etal., supra), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also include a liposomally associatedcompound. Liposomes containing the compound are prepared by methodsknown to those of skill in the art, as exemplified by DE 3,218,121;Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appln. 83-118008, U.S. Pat. Nos. 4,485,045 and4,544,545 and EP 102,324. In some embodiments, liposomes are of thesmall (from or about 200 to 800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the most efficacious therapy. AllU.S. parents referred to herein, both supra and infra, are herebyexpressly incorporated by reference in their entirety.

PEGylated peptides having a longer life than non-PEGylated peptides canalso be employed, based on, for example, the conjugate technologydescribed in WO 95/32003 published Nov. 30, 1995.

In some embodiments, the compound can be formulated generally by mixingeach at a desired degree of purity, in a unit dosage injectable form(solution, suspension, or emulsion), with a pharmaceutically, orparenterally, acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides. It can be appreciated thatthe above doses are not intended to be limiting. Other doses outside theabove ranges can be determined by those with skill in the art.

In some embodiments, formulations can be prepared by contacting acompound uniformly and intimately with liquid carriers or finely dividedsolid carriers or both. Then, if desired, the product can be shaped intothe desired formulation. In some embodiments, the carrier is aparenteral carrier, alternatively, a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, a buffered solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity, and chemical stability. Suchmaterials are desirably non-toxic to recipients at the dosages andconcentrations employed, and include, by way of example only, bufferssuch as phosphate, citrate, succinate, acetic acid, and other organicacids or their salts; antioxidants such as ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;glycine; amino acids such as glutamic acid, aspartic acid, histidine, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, mannose, trehalose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; counter-ions such as sodium; non-ionic surfactants such aspolysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutralsalts, e.g., NaCl, KCl, MgCl₂, CaCl₂, and the like. In certainembodiments, a peptide of this invention can be stabilized using 0.5 Msucrose or 0.5 M trehalose. Using such sugars can permit long-termstorage of the peptides.

An NRP compound can be desirably formulated in such vehicles at a pH offrom about 6.5 to about 8. Alternatively, the pH can be from about 4.5to about 8. It will be understood that use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofsalts of the compound. The final preparation may be a stable liquid orlyophilized solid.

In other embodiments, adjuvants can be used. Typical adjuvants which maybe incorporated into tablets, capsules, and the Iike are a binder suchas acacia, corn starch, or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent like corn starch oralginic acid; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose or lactose; a flavoring agent such as peppermint,wintergreen, or cherry. When the dosage form is a capsule, in additionto the above materials, it may also contain a liquid carrier such as afatty oil. Other materials of various types may be used as coatings oras modifiers of the physical form of the dosage unit. A syrup or elixirmay contain the active compound, a sweetener such as sucrose,preservatives like propyl paraben, a coloring agent, and a flavoringagent such as cherry. Sterile compositions for injection can beformulated according to conventional pharmaceutical practice. Forexample, dissolution or suspension of the active compound in a vehiclesuch as water or naturally occurring vegetable oil like sesame, peanut,or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants, and the likecan be incorporated according to accepted pharmaceutical practice.

Desirably, an NRP analogue to be used for therapeutic administration maybe sterile. Sterility can be readily accomplished by filtration throughsterile filtration membranes (e.g., membranes having pore size of about0.2 micron). Therapeutic compositions generally can be placed into acontainer having a sterile access port, for example an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In other embodiments, an NRP analogue can be stored in unit ormulti-dose containers, for example, sealed ampules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-mL vials are filled with 5ml of sterile-filtered 0.01% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution can be preparedby reconstituting lyophilized compounds using bacteriostatic water orother suitable solvent.

In still further embodiments, a kit may contain a predetermined amountof lyophilized NRP compound, a physiologically compatible solution forpreparation of a dosage form, a mixing vial, a mixing device, andinstructions for use. Such kits can be manufactured and stored accordingto usual practices in the industry.

An NRP compound-containing composition may be administered by one ormore of a variety of routes. By way of example, intravenous,intraperitoneal, intracerebral, intraventricular, inhalation, lavage,rectal, vaginal, transdermal, subcutaneous administration can be used.

EXAMPLES

The following examples are presented to illustrate specific embodimentsof this invention. Persons of ordinary skill can utilize the disclosuresand teachings herein to produce other embodiments and variations withoutundue experimentation. All such embodiments and variations areconsidered to be part of this invention.

Example 1 Effects of NRP Compounds on Survival and Proliferation ofCerebellar Microexplants

NRP Compound Preparation

NRP compounds were provided by Auspep (Australia). The peptides weresynthesized using standard solid-phase synthesis. The peptides weresupplied with an amidated C-terminus, and were more than 95% pure asanalyzed by MALDI-MS spectrum analysis. The peptides were storedlyophilized at −80° C. under argon in 0.5M sucrose or 0.5M trehaloseuntil usage. They were reconstituted in PBS, alternatively in 100 μg/mlhuman transferrin/PBS or in other embodiments in 100 μg/ml BSA/PBS, in0.5M sucrose or 0.5M trehalose.

Cell Culture Preparation

Laminated cerebellar cortices of the two hemispheres were explanted froma P3, P4, P7 or P8 Wistar rat, cut into small pieces in GBSS with 0.65%D(+)glucose solution, and triturated by a 0.4 mm gauge needle andsubsequently pressed through a 125 μm pore size sieve. The obtainedmicroexplants were centrifuged (60×g) 2 times for a medium exchange intoserum-free BSA-supplemented START V-medium (Biochrom). Finally, themicroexplants were reconstituted in 500 μl STARTV-medium. For culturing,38 μl of the cell suspension was incubated for 1 hour on apoly-D-lysine-coated cover slip in a 35 mm Petri dish under anatmosphere comprising 5% CO₂ in air and 100% humidity at 34° C.Subsequently, the injuring toxins (as described below), NRP analoguesand 1 ml of STARTV-medium were added, and the cultures were evaluatedafter 2-3 days of culture.

For immunohistochemistry and neuronal migration experiments, cerebellarmicroexplants were fixed after 2-3 days in culture after the followingregime: microexplants were fixed by 2-minute, serial treatment with0.4%; 1.2%; 3% paraformaldehyde, respectively, followed by a 5 minincubation in 4% paraformaldehyde/0.25% glutaraldehyde in 0.1 M sodiumphosphate (pH 7.4).

Effects of NRP Compounds on Toxin-Induced Neural Injury

Oxidative stress can result in neurodegeneration. This is one possiblemechanism for the symptoms observed in human disorder with Huntington'sdisease. The oxidative stress-producing toxin, 3-nitropropioinic acid(3-NP) has been previously shown to produce effects in experimentalanimals that mimic those effects seen in human beings with Huntington'sdisease. Thus, studies of therapeutic drugs in experimental animals with3-NP induced neurotoxicity are predictive of effects of those drugs intreating human beings with Huntington's disease or other disorderscharacterized by oxidative stress.

General methods for toxicological and drug administration experimentswere designed such that 1/100 parts of toxin and neuroprotective drugwere administered simultaneously to the freshly prepared cerebellarmicroexplants. Glutamate was prepared as a 50 mM stock solution inMilliQ water while 50 mM 3-nitropropionic acid (3-NP) was pH-adjusted(pH 6.8-7.2) in MilliQ water. The concentrations of the oxidative stressinducing toxin, 3-nitropropionic acid (3-NP), and the excitotoxin,glutamate, in the assay were at concentrations of 0.5 mM each.Lyophilized NRP peptides were reconstituted in PBS or 100 μg/ml humantransferrin as a 10 μM stock solution. Subsequently, serial dilutionswere made. Cerebellar microexplants were cultivated for 48-72 hours at34° C., 5% CO₂ in air and 100% humidity before they were fixed byincreasing amounts of paraformaldehyde (0.4%, 1.2%, 3% and 4%—eachtreatment 2-3 min).

Using the toxins described above, cerebellar explants were exposed for24 hours, at the beginning of culturing to dilutions of NRP (survivalassay) or NRP and 0.1 μM BrdU (proliferation assay). Subsequently, 80%of the medium was changed without addition of new toxins and NRPs. Thecerebellar cultures were fixed as described above after 3 days in vitro.The detection of the incorporated BrdU level is performed as describedpreviously.

Data Reduction and Statistical Analysis

For statistical analysis of survival, four fields (each field having anarea of 0.65 mm²) of each fixed cerebellar culture with the highest celldensities were chosen, and cells displaying neurite outgrowth werecounted (survival assay).

Results

Neuroprotection

NRP analogues promoted increased neuronal survival in explants treatedwith 3-NP (see Examples 2, 3 and 4).

Example 2 Promotion of Neural Cell Survival by NRP Compounds

We studied effects of different concentrations of SEQ ID NO:1, and SEQID NO:5 of this invention on cell cultures prepared according to Example1 above. SEQ ID NO:1 and SEQ ID NO:5 have the same amino acid sequence,with the exception that the Alanine (A) in position 9 of the amino acidsequence of SEQ ID NO:1 was replaced by aminoisobutyric acid (Aib), thusproducing a compound having the sequence shown in SEQ ID NO:5. One canappreciate that in addition to the change in linear structure of thepeptide, Aib replacement of Alanine can stabilize the beta-turn, andthus, produce a peptide having different 3-dimensional structure anddifferent mobilities about certain bonds compared to the un-substitutedpeptide.

We exposed cell cultures to vehicle alone (open column) the neurotoxin3-NP alone (shaded column) to 3-NP plus different concentrations of SEQID NO:5 (hatched columns) or to 3-NP plus different concentrations of anon-substituted NRP, SEQ ID NO:1 (dark shaded column). We then countedthe numbers of cells having neurites as an indicator of neuronal cellsurvival.

FIG. 1 depicts a graph of results of these studies. 3-NP alone producedprofound loss of cells displaying neurites compared to vehicle-treatedcontrols, indicating that the compound is indeed neurotoxic. The peptidehaving the sequence SEQ ID NO:1 at concentrations of 10 fM or 1 pMsignificantly decreased the neurotoxic effects of 3-NP (mean±SEM;p<0.001; n=4). Similarly, SEQ ID NO:5 decreased 3-NP-inducedneurotoxicity in a concentration dependent fashion, with a threshold ofbelow about 1 fM and a maximal effect at a concentration of about 100 fM(mean±SEM; p<0.001; n=4 each).

From this study, we conclude that SEQ ID NO:5 is neuroprotective. Thisresult means that SEQ ID NO:5 can be a valuable therapeutic NRP compoundfor treating neural degeneration in humans suffering from neurologicaldisorders. Further, because oxidative stress is known to induceneurodegeneration similar to the neurodegeneration observed in humanbeings with Huntington's disease, synthetic NRP compounds of thisinvention can be used to treat human beings with neurodegenerationcaused by oxidative stress.

Example 3 Stability and Neuroprotective Effects of Un-Substituted NRPCompounds

To determine the stability of NRPs of this invention in storage, wecarried out a series of studies using SEQ ID NO:1. In this study, wesynthesized SEQ ID NO:1 and then stored the peptide at temperatures ofeither −20° C. or −4° C. for nine (9) weeks. We then tested the NRP forefficacy in protecting cerebellar neurons against the neurotoxic effectof 3-NP as described above. Cerebellar explants were treated withvehicle alone (open column), the neurotoxic agent 3-NP alone (lightstippled column), or 3-NP plus four concentrations of SEQ ID NO:1 thathad been stored for 9 weeks at −20° C., or at −4° C.

FIG. 2 depicts results of these studies. Cerebellar explants treatedwith 3-NP alone (light stippled bar) showed fewer cells exhibitingneurites compared to vehicle-treated control explants (open bar). SEQ IDNO:1 that had been stored at −20° C. exhibited neuroprotective effectsat all concentrations tested (from 10⁻¹³ M; 100 fM to 10⁻¹⁰ M; 100 pM),with statistically significant effects observed at concentrations of10⁻¹¹ M and 10⁻¹⁰ M.

In contrast, SEQ ID NO:1 stored at a temperature of −4° C. producedlittle decrease in neurotoxic effect of 3-NP. We conclude from thisstudy that the unsubstituted NRP loses activity over the 9-week periodwith storage at −4° C., and that storing SEQ ID NO:1 at −20° C. canprotect its potency.

Example 4 Stability and Neuroprotective Effects of Substituted NRPs

In this study, we determined the stability of a substituted NRP, SEQ IDNO:5, in different storage conditions and at different concentrations aswith Example 3 above. We synthesized SEQ ID NO:5 and then stored thepeptide for 9 weeks at a temperature of either −20° C. or −4° C. We thentested the efficacy of SEQ ID NO:5 in decreasing, the neurotoxic effectof 3-NP as described in Examples 2 and 3 above.

FIG. 3 depicts a graph of these studies. Cerebellar explants weretreated with vehicle alone (open column), 3-NP alone (light stippledcolumn) or 3-NP plus concentrations of SEQ ID NO:5 in fourconcentrations ranging from 10⁻¹³ M to 10⁻¹⁰ M) that had been stored ateither −20° C. or at −4° C. When stored at −20° C., SEQ ID NO:5 producedneuroprotective effects similar to those found for SEQ ID NO:1 that hadbeen stored at −20° C., as shown in Example 3 and depicted in FIG. 2.

We surprisingly found that even when stored at −4° C., SEQ ID NO:5retained its neuroprotective effect. In fact, the degree ofneuroprotection provided by SEQ ID NO:5 after storage at −4° C. was notstatistically different from the degree of neuroprotection providedafter storage at −20° C. This result was completely unexpected based onstudies of SEQ ID NO:1 shown above. The increased stability of the NRPhaving SEQ ID NO:5 means that this compound will be more suitable forstorage and transportation under commonly used conditions.

Example 5 Neuroprotective Effects of Un-Substituted and Substituted NRPs

In a larger set of data obtained using the methods of Example 1, weconfirmed the results shown in FIG. 1. FIG. 4 depicts a graph of resultsof studies of cerebellar microexplants treated with the neurotoxin 3-NP,demonstrating neuroprotection by SEQ ID NO:5 and SEQ ID NO:1 in a studyof 6 in each group. We conclude that SEQ ID NO:1 and SEQ ID NO:5 protectneurons from dying after exposure to the neurotoxin 3-NP. We alsoconclude that SEQ ID NO:5 and SEQ ID NO:1 can be useful therapeuticagents in treating human beings suffering from neurotoxicity.

Example 6 Therapeutic and Prophylactic Effects of NRP Analogues in aModel of Multiple Sclerosis

To determine whether NRPs have an impact on chronic inflammation in theCNS that leads to severe axonal damage and subsequent lesions (such asin multiple sclerosis; MS), we tested NRPs in a mouse model ofexperimental autoimmune encephalitis (EAE) that mimics the severeprogressive state of MS, using myelin oligodendrocyte glycoprotein (MOG)as the immunogen.

Methods and Materials

Animals

Female Mice, 6-8 weeks-old, strain C57Bl/6J weighing an average of 24gms each were used.

NRP Preparation

The peptide having the sequence:

NH₂-REGRRDAPGRAGG-NH₂ SEQ ID NO: 12(also known as SEQ ID NO: 30 disclosed in U.S. patent application Ser.No. 10/976,699), was supplied by Auspep (Australia). The peptide SEQ IDNO:12 was supplied with an amidated C-terminus, and was more than 95%pure as determined by MALDI-MS spectroscopy.HPLC. The sequence wasconfirmed by mass spectroscopy. The peptide was stored lyophilized at atemperature of −80° C. under argon gas until use. The peptide wasreconstituted in PBS on the day of use.

Induction EAE

A 200 μul L of an emulsion containing 200 μug of the encephalitogenicpeptide MOG35-55

MEVGWYRSPFSRVVHLYRNGK SEQ ID NO: 13was obtained from C S Bio Co. USA) in complete Freund adjuvant (Difco,Detroit, USA) containing 800 μug Mycobacterium tuberculosis (Difco,Detroit, USA) was injected subcutaneously into one flank. Mice wereimmediately injected intraperitoneally with 400 ng pertussis toxin (ListBiological Laboratories, USA) and again 48 hours later.

Treatment

Therapeutic

At the peak of the disease (day 17 after MOG-immunization) animals weretreated with SEQ ID NO: 12 intraperitoneally (i.p.) for 14 days with adaily dose of 0.1 μg peptide/animal (4.16 μg/kg).

Assessment of Neurological Impairment

Mice were monitored daily and neurological impairment was scored on anarbitrary clinical score as follows: 0, no clinical sign; 1, flaccidtail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limbweakness and fore limb weakness; 5, paraplegia; 6, death.

Results

Therapeutic Effects of NRPs on EAE in Mice

The outcome 37 days after the first NRP treatment is shown in FIG. 5.There is a significant therapeutic effect of SEQ ID NO: 12 whenperipherally administered. A similar drug effect has been shown for theneuroregenerative compound EPO in a combination therapy withmethylprednisolone. The disadvantage of EPO is its large size as itcannot easily be synthesized or administered. We conclude that NRP hassignificant long-term potential to decrease the severity of motoricimpairment occurring in the EAE disease model of MS when administered astherapeutic drug at the peak of the disease. Score 1 is the lowest scoreand implies a flaccid tail only while the higher scores imply weakness(score 2) or complete paralysis of the hind legs (score 3). **p<0.01versus treatment day 1 score.

Example 7 Effects of NRP Analogues in Animals with Peripheral Neuropathy

To determine if NRP analogues of this invention can be usefultherapeutic agents for treating peripheral neuropathy, we carried out aseries of studies in rats with peripheral neuropathy induced bypyridoxine.

We demonstrated that NH₂-G¹RRAAPGR-Aib-GG¹¹-NH₂ (SEQ ED NO:5) at verylow doses applied as a single bolus per day can attenuate motor deficitsin animals treated with toxic doses of pyridoxine.

Materials and Methods

Male Sprague-Dawley rats were used and were weighed 278-349 g at thecommencement of intoxication with pyridoxine chloride. Beforeintoxication, rats were habituated to walk across a beam at dailyintervals for a week.

Experiment I: Rats were administered 400 mg/kg pyridoxine chloridedissolved in sterile distilled water adjusted to neutral pHintraperitoneally (i.p) twice daily for 8 days and concurrently thepeptide having the sequence SEQ ID NO:5 was administered i.p. for attotal of 10 days. The rats were observed for a total of 29 days.

Experiment II: Rats were administered a higher dose of pyridoxine (1200mg/kg/day) over a shorter period of time. Pyridoxine chloride dissolvedin sterile distilled water adjusted to neutral pH was administered i.p.to the rats at a dose of 600 mg/kg twice daily for 4 days. Concurrentlythe peptide having the sequence SEQ ID NO:5 was administered for 4 days.On day 5, rats were tested for motor deficits.

Motor deficits after pyridoxine intoxication and effects of SEQ ID NO:5were analyzed using a precision beam walk. Seven subsequent foot stepsacross the 1.5 m long beam were videotaped and according to thepositioning of the foot tarsus, these steps were scored between 1 to 4(1—hind leg tarsus above the beam median; 2—tarsus was touching theupper half of beam medium; 3—tarsus was touching the lower half of beammedian and 4—tarsus below the beam median). The score results of allseven steps were added together. A score of 30 was given if the animalwas only able to stand on the beam but was unable to walk. In the eventof inability to stand on the beam a score of 32 was awarded.

Treatment Groups

Experiment I: The two concentrations tested for SEQ ID NO:5 were 40ng/kg/day and 4 μg/kg/day.

Experiment II: Saline and SEQ ID NO:5 at a dose of 4 μg/kg/day weretested.

Statistics Analysis

Motor deficit data were assessed by two-way analysis of variance andBonferroni post-hoc test. Statistical significance was concluded ifp<0.05 between drug treatment cohorts and vehicle treatment.

Results

Experiment I:

Four days after cessation of pyridoxine-intoxication both SEQ IDNO:5-treated rat cohorts show attenuated motor deficits (low dosage:p<0.05; high dosage: p<0.01) compared to the vehicle-treated group. Atday 16 after start of pyridoxine-intoxication the high dose SEQ ID NO:5group had still significantly less motor deficits than the control group(p<0.05). (FIG. 6).

Experiment II:

Four subsequent days of high-dosage pyridoxine-intoxication lead tosubstantial motor deficits in the control group of rats. On day 5 theSEQ ID NO:5-treated rat group (dosage: 4 μg/kg/day) showed highlysignificantly attenuation of motor deficits (p<0.001) (FIG. 7).

Conclusions

We conclude that SEQ ID NO:5 showed highly significant and clinicallysubstantial attenuation of motor deficits pyridoxine-induced peripheralneuropathy. We also conclude that at a dose of 4 μg/kg/day, SEQ ID NO:5was well tolerated. The 4 μm/kg/day concentration worked equally well inthe more chronic model of pyridoxine intoxication (800 mg/kg/day for 8days) and the acute pyridoxine intoxication induced by 1200 mg/kg/dayadministered for 4 days.

We further conclude from these studies that SEQ ID NO:5 can be effectivetreatment for peripheral neuropathies in human beings.

Example 8 Effect of SEQ ID NO:5 on Animals with Amytrophic LateralSclerosis (ALS)

In this series of studies, we examined the effects of SEQ ID NO:5 onmice having a genetic defect (SOD-1) that produces a motor neurondisease similar to that in humans with ALS. Animals with this disordershow a progressive loss of motor coordination over time, and ultimatelydie prematurely of the disease. This animal system is useful in studyingeffects of agents potentially useful in treating ALS in human beings.Thus, results obtained are highly predictive of results obtained inhumans with ALS.

Methods

Mice were randomly allocated to receive either vehicle or SEQ ID NO:5from the point of disease onset onwards. Disease onset in each treatmentallocation group was not significantly different: 92-93 days.

SEQ ID NO:5 treatment (40 μg/kg, given 1/day, i.p. (FIG. 8A) or 0.4μg/kg, given 1/day, i.p. (FIG. 8B)) was started at the day of onset ofthe disease.

Results

In two studies, SEQ ID NO:5 treatment resulted in a significantextension of lifetime in the mice suffering from the ALS-like disorder(FIGS. 8A and 8B). A daily dose of 40 μg/kg i.p. SEQ ID NO:5significantly promoted longevity following disease onset. FIGS. 8A and8B show Kaplan-Meier survival probability curves for the SOD-1 mutant(ALS) transgenic mice.

The vehicle-treated animals began to die at day 120, and were all deadby day 143 (Study 1; FIG. 8A; solid line) Similarly, in Study 2 (FIG.8B; solid line), vehicle-treated animals began to die at day 120 asnwere all dead at day 154 (FIG. 8B; solid line).

In contrast, in Study 1 (FIG. 8A; dashed line) SEQ ID NO:5-treated micebegan dying later (at day 131) than vehicle-treated animals and livedlonger (up to 156 days) Significantly, the two Kaplan-Meier curves didnot overlap (FIG. 8A; 40 μg/kg SEQ ID NO:5; p<0.01 compared to survivalof animals treated with vehicle alone). In Study 2 animals treated withSEQ ID NO:5 (FIG. 8B; dashed line; 0.4 μg/kg) began to die later (at day125) than vehicle-treated animals (FIG. 8B; solid line) and in general,lived longer (up to 189 days: FIG. 8B; dashed line; SEQ ID NO:5.

Conclusions

We conclude from these studies that SEQ ID NO:5 is an effective agentthat has unexpectedly improved stability compared to its un-substitutedcounterpart. We also conclude that stabilizing the beta-turn of an NRPcan improve therapeutic efficacy and can improve stability, both ofwhich can improve therapeutic utility of NRP analogues. The unexpectedfindings that substituted NRP analogues have both improved potency andimproved stability indicates that the NRP analogues can be valuabletherapeutic alternatives for treating a variety of conditionscharacterized by neurodegeneration.

Therefore, NRP analogues of this invention can be useful in treatingacute as well as chronic neurodegenerative disorders including ALS,neurotoxicity, neurodegeneration associated with oxidative stress,autoimmune disorders, traumatic brain injury and other neurologicaldiseases and conditions. Further, we conclude that NRP analogues canhave beneficial therapeutic effects in situations where loss ofneurological function is a symptom.

Example 9 NRP Analogue-Mediated Migration in Physiological (Injury-Free)Conditions

An NRP analogue is tested for migration-inducing/chemoattractiveactivity on mouse neural stem cells in a haptotactic migration assay asdescribed below.

Methods

Initial NRP Coating

Control wells of Transwell plates (Corning) with 12 μm pore size arecoated in 1.5 ml of the BSA/PBS vehicle. Remaining plates are coatedusing 0.1 ng/ml of NRP analogue (prepared in PBS containing 10 ug/mlBSA).

Extracellular Matrix Coating

Laminin (7 μg/ml) is used as extracellular matrix (ECM) coating formouse primary stem cells. The matrix is incubated at 37° C.; 5% CO₂ for2 hrs at room temperature. The cells are seeded onto the inserts (30,000cells per well). Plates are fixed at 1-2 days in vitro (DIV).

Coating of Inserts

A 5 ug/mL PDL/PLL mixture (in PBS) is used to coat inserts. Subsequentlythe inserts are rinsed with MilliQ water.

Cell Fixation

Inserts are discarded and wells fixed in successive dilutions of PFA(0.4, 1.2, 3 and 4%) for 3-5 min in each dilution. The wells are rinsedand stored in successive dilutions of PFA (0.4, 1.2, 3 and 4%) 3-5 minin each dilution. The wells are rinsed and stored in PBS until counting.All cells that display neurite outgrowth and traveled to the bottomchamber are counted as migrating cells.

Results

More cells migrate in plates treated with NRP analogue than migrated inplates without NRP analogue. NRP analogues can induce neuronal cellmigration, and that they each can be used to treat neurodegenerationassociated with neural injury or disease.

Example 10 NRP Analogue-Mediated Migration in Injury Conditions

An NRP analogue is tested for migration-inducing/chemoattractiveactivity on mouse neural stem cells in a haptotactic migration assay ininjury conditions, as described below.

Methods

Production of a Monolayer of Astrocytes

P1 (postnatal day 1) Wistar or Sprague Dawley rats are sacrificed bydecapitation. Cortical heminspheres are removed and collected intoseparate tubes containing 4 ml DMEM—1 cortex per tube. The tissue ismechanically triturated. Cells are transferred into medium using asterile pipette and filtered through a 100 um cell strainer into a 50 mlcentrifuge tube. Each tube is stocked up to 50 ml with DMEM. The tubesare centrifuged for 5 mins at 350×g at 22° C. The cells are resuspendedin 40 ml of DMEM+10% FBS. The cells are then seeded into a 12-wellplate+5 nM ocadaic acid (to remove neurons by inducing apoptotic celldeath) and incubated at 37° C./10% CO₂ for 24 hrs in a Boyden Chamber.The medium+FBS is replaced after 1 day with fresh DMEM+10% FBS. The cellgrowth is monitored until confluency (14-18 days).

Pharmacologocal and Mechanical Injury.

Induction of injuries to the astrocytic monolayer is accomplished usingthe pharmacological agent transforming growth factor β1 (TGFβ1) andsimultaneous mechanical scratching of the monolayer in order to activateastrocytics. 10 ng/ml TGFβ1 is administered to the astrocytic monolayerfor 24 hrs. Additionally, astrocytic cultures re mechanically injured bya scalpel (one scratch throughout the bottom of the well).

Seeding of Pre-Labelled Stem Cells

Undifferentiated fluorescein diacetate-labelled embryonic mouse neuralstem cells (NSCs) are seeded into Poly-D-Lysine (PDL—5 μg/ml) coatedinserts. The lower compartment of the Boyden chamber receives 100 fM ofan NRP analogue.

Cell Fixation

Inserts are discarded and wells fixed in successive dilutions of PFA(0.4, 1.2, 3 and 4%) for 3-5 min in each dilution. The wells are rinsedand stored in successive dilutions of PFA (0.4, 1.2, 3 and 4%) 3-5 minin each dilution. The wells are rinsed and stored in PBS until counting.All cells that display neurite outgrowth and traveled to the bottomchamber are counted as migrating cells.

Analysis

Migrated stem cell number of labelled cells re analysed after 24 hrs bya fluorescence-based computerized imaging system (Discovery-1).

Results

NRP analogues stimulate more stem cells to migrate than vehicle-treatedcontrols. We conclude that NRP analogue induces neuronal stem cellmigration, and that NRP analogues can be useful to treatneurodegeneration associated with neural injury or disease.

REFERENCES

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All patents, patent applications and other publications are incorporatedherein fully by reference as if separately so incorporated. The SequenceListing appended to this application is also incorporated herein fullyby reference.

A person of ordinary skill in the art will not have to undertake undueexperimentation, taking account of that skill and the knowledgeavailable, and of this disclosure, in developing one or more suitablesynthetic compounds. All such compounds and methods for theirmanufacture and use are considered to be part of this invention.

Compounds and compositions of this invention find industrial use in manyaspects of commerce, including pharmaceutical manufacturing,formulation, and sale. Methods of using compounds and compositions ofthis invention find industrial use in medical fields of neurology, andin particular, for treatment of neurological disorders in animals andhuman beings.

1. A synthetic neural regeneration peptide (NRP) compound comprising theamino acid sequence selected from the group consisting of SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6.
 2. The synthetic NRP compound of claim 1consisting of the amino acid sequence of SEQ ID NO:5.
 3. Apharmaceutical composition comprising a synthetic NRP compound of claim1 or claim 2 and a pharmaceutically acceptable excipient.